This application will focus on the critical pre-clinical experiments required to advance a novel antibiotic peptide into clinical trials. We successfully synthesized a twenty-five amino acid bioactive peptide (CAP372o-44) based on the native sequence of the natural host defense molecule known as CAP37. CAP372o-44 and two peptide analogs, CAP372o-44Ser26 and CAP372o-44 Ser42, have potent antimicrobial activity for Gram-negative bacteria, including Pseudomonas aeruginosa and Acinetobacter baumannii. Of additional importance to the antibiotic activity of CAP37 peptides, is their ability to bind and neutralize the toxic effects of lipopolysaccharide (LPS) or endotoxin, a component of the outer membrane of Gram-negative organisms. We hypothesize that CAP37 peptides are a new class of antibiotic, and due to their novel mode of antimicrobial activity and LPS binding activity, will prove to be effective therapies for drug-resistant healthcare-associated infections. Our goal is the development of a novel antibiotic for the treatment of severe Gram-negative infections in an acute hospital setting. Our initial indications are for the treatment of bacteremia and nosocomial pneumonia due to P. aeruginosa. This application is structured to satisfy the key milestones required for successful submission of an investigational new drug application to the FDA. The application has five major areas of study and development which include 1) selection of lead compound, 2) production and formulation, 3) in vitro microbiology, 4) advanced disease modeling studies to establish in vivo efficacy, and 5) pharmacokinetics, toxicology, and safety pharmacology. Aim 1: To establish the scale-up procedure of the synthesis of CAP37 peptides to ensure sufficient GMP-like compound for experimental analysis and to determine feasibility of cost per gram at scale for use in clinical trials. Aim 2: To determine the in vitro efficacy and antimicrobial spectrum of activity of the CAP37 peptides using Clinical and Laboratory Standards Institute (CLSI) reference methods and to examine the propensity for organisms to develop resistance to CAP37 peptides via spontaneous and sequential mutation. Aim 3: To determine the in vivo efficacy of CAP37 peptides in rodent and nonhuman primate models of Pseudomonas pneumonia and bacteremia. Aim 4: To determine the pharmacokinetics of the peptides in two animal species and to perform the required safety pharmacology and toxicology to meet FDA requirements for an IND submission. This application will provide key insights into the mechanism of action of a novel antibiotic for treating multiple severe Gram-negative infections. Relevance to public health. There is a critical need for new antibiotics to treat health-care associated infections. The increased incidence of pathogen resistance to current antibiotics results in dramatically increased morbidity and mortality rates, a serious public health issue. Identifying new therapeutics with strong antimicrobial activity and low propensity to induce resistance will be of great public health importance.